Steroidobacter Denitrificans Research Articles

Key genes of electron transfer, the nitrogen cycle and tetracycline removal in bioelectrochemical systems

BackgroundSoil microbial fuel cells (MFCs) can remove antibiotics and antibiotic resistance genes (ARGs) simultaneously, but their removal mechanism is unclear. In this study, metagenomic analysis was employed to reveal the functional genes involved in degradation, electron transfer and the nitrogen cycle in the soil MFC.ResultsThe results showed that the soil MFC effectively removed tetracycline in the overlapping area of the cathode and anode, which was 64% higher than that of the control. The ARGs abundance increased by 14% after tetracycline was added (54% of the amplified ARGs belonged to efflux pump genes), while the abundance decreased by 17% in the soil MFC. Five potential degraders of tetracycline were identified, especially the species Phenylobacterium zucineum, which could secrete the 4-hydroxyacetophenone monooxygenase encoded by EC 1.14.13.84 to catalyse deacylation or decarboxylation. Bacillus, Geobacter, Anaerolinea, Gemmatirosa kalamazoonesis and Steroidobacter denitrificans since ubiquinone reductase (encoded by EC 1.6.5.3), succinate dehydrogenase (EC 1.3.5.1), Coenzyme Q-cytochrome c reductase (EC 1.10.2.2), cytochrome-c oxidase (EC 1.9.3.1) and electron transfer flavoprotein-ubiquinone oxidoreductase (EC 1.5.5.1) served as complexes I, II, III, IV and ubiquinone, respectively, to accelerate electron transfer. Additionally, nitrogen metabolism-related gene abundance increased by 16% to support the microbial efficacy in the soil MFC, and especially EC 1.7.5.1, and coding the mutual conversion between nitrite and nitrate was obviously improved.ConclusionsThe soil MFC promoted functional bacterial growth, increased functional gene abundance (including nitrogen cycling, electron transfer, and biodegradation), and facilitated antibiotic and ARG removal. Therefore, soil MFCs have expansive prospects in the remediation of antibiotic-contaminated soil. This study provides insight into the biodegradation mechanism at the gene level in soil bioelectrochemical remediation.

Electrogenic engineered flow through tri-phasic wetland system for azo dye treatment: Microbial dynamics and functional metagenomics

Electrogenic engineered flow through tri-phasic wetland (EEFW) system based on nature-based ecological principles was studied by integrating successive biological microenvironments. The potential mechanism of the plant root-based microbial community and its functional diversity with the influence of plant-microbe-electrode synergism towards dye degradation was evaluated. The EEFW system was operated at three varied dye loads of 10, 25 and 50 mg L−1, where the results from the cumulative outlets revealed a maximum dye removal efficiency of 96%, 96.5% and 93%, respectively. Microbial community analysis depicted synergistic dependence on the plant-microbe-electrode interactions, influencing their functional diversity and metabolism towards detoxification of pollutants. The core microbial taxa enriched against the microenvironment variation were mostly associated with carbon and dye removal viz., Desulfomonile tiedjei and Rhodopseudomonas palustris in Tank 1 and Chloroflexi bacterium and Steroidobacter denitrificans in Tank 2. The degradation of polycyclic aromatic hydrocarbons, chloroalkane/chloroalkene, nitrotoluene, bisphenol, caprolactam and 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) were observed to be predominant in Tank 1. EEFW system could be one of the option for utilizing nature-based processes for the treatment of wastewater by self-induced bioelectrogenesis to augment process efficiency.

Random forest analysis reveals taxa predictive of Prunus replant disease in peach root microbiomes

Successive plantings of Prunus species produce suboptimal growth and yield in many California soils due to a poorly understood soilborne disease complex, Prunus replant disease (PRD). We explored the hypothesis that PRD is mediated by microbial taxa in roots of Nemaguard peach, a rootstock for almond and other stone fruits. In a greenhouse bioassay, portions of 10 replant soils were treated with fumigation or pasteurization or left untreated as a control before being planted with peach seedlings. Ten weeks after planting, seedlings were considered PRD-affected if their top fresh weights in the control were significantly reduced, compared to the weights in pasteurization and fumigation treatments; plants with equivalent top weights in all treatments were considered to be non-affected. The roots were washed from the soil, frozen, extracted for total DNA, and used for metabarcoding of rRNA gene amplicons from bacteria, fungi, and oomycetes. High-throughput amplicon sequencing revealed that root microbial community shifts resulted from preplant treatments, and specific taxa were associated with PRD induction among controls. Random forest (RF) analysis discriminated effectively between PRD-affected and non-affected root communities. Among the 30 RF top-ranked amplicon sequence variant (ASV) predictors, 26 were bacteria, two were oomycetes, and two were fungi. Among them, only Streptomyces scabiei, Steroidobacter denitrificans, Streptomyces bobili, and Pythium mamillatum had root abundances ≥5% that were either associated positively (former two ASVs) or negatively (latter two) with PRD. Thus, our findings were consistent with microbial mediation of PRD in roots and suggested taxa that may be involved in the mediation.

Metagenomic analysis reveals functional genes in soil microbial electrochemical removal of tetracycline

Microbial fuel cells (MFCs) are capable of removing tetracycline in soils, in which the degradation efficiency of tetracycline is hindered by its strong adsorption capacity. Phosphate was chosen as a competitor for tetracycline adsorption to improve its removal rate in soil MFCs. The results showed that 42–50% of tetracycline was degraded within 7 days, which was 42–67% higher than open-circuit treatments. Compared with closed-circuit treatments without phosphate addition, the removal efficiencies of tetracycline after phosphate addition increased by 19–25% on day 51, and accumulated charge outputs were enhanced by 31–52%, while the abundance of antibiotic resistance genes decreased by 19–27%. Like Geobacter, the abundance of Desulfurispora and Anaeroomyxobacter in the anode showed similar tendencies with current densities, suggesting their dominant roles in bioelectricity generation. Gemmatimonadetes bacterium SCN 70–22, Azohydromonas australica, Steroidobacter denitrificans and Gemmatirosa kalamazoonesis were found to be potential electrotrophic microbes in the cathode. The expressed flavoprotein 2,3-oxidoreductase, quinol oxidase and fumarate reductase might have promoted the transfer efficiency of electrons from cathodes to cells, which finally accelerated the biodegradation rate of tetracycline in addition to the polyphenol oxidase. This study provides an insight into functional enzyme genes in the soil microbial electrochemical remediation.

Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment.

Steroid hormones, such as androgens, are common surface-water contaminants. However, literature on the ecophysiological relevance of steroid-degrading organisms in the environment, particularly in anoxic ecosystems, is extremely limited. We previously reported that Steroidobacter denitrificans anaerobically degrades androgens through the 2,3-seco pathway. In this study, the genome of Sdo. denitrificans was completely sequenced. Transcriptomic data revealed gene clusters that were distinctly expressed during anaerobic growth on testosterone. We isolated and characterized the bifunctional 1-testosterone hydratase/dehydrogenase, which is essential for anaerobic degradation of steroid A-ring. Because of apparent substrate preference of this molybdoenzyme, corresponding genes, along with the signature metabolites of the 2,3-seco pathway, were used as biomarkers to investigate androgen biodegradation in the largest sewage treatment plant in Taipei, Taiwan. Androgen metabolite analysis indicated that denitrifying bacteria in anoxic sewage use the 2,3-seco pathway to degrade androgens. Metagenomic analysis and PCR-based functional assays showed androgen degradation in anoxic sewage by Thauera spp. through the action of 1-testosterone hydratase/dehydrogenase. Our integrative 'omics' approach can be used for culture-independent investigations of the microbial degradation of structurally complex compounds where isotope-labeled substrates are not easily available.

The Growth Inhibition Effect on the Bacterial Vaginosis Causative Bacteria by Citric Acid and Trisodium Phosphate

Bacterial vaginosis (BV) is the most frequent vaginal disease being apt to relapse. The growth inhibition effect of the mixture of citric acid (CA) and trisodium phosphate (TSP) on BV causative bacteria and probiotics was measured.Gardnerella vaginalis was reduced to zero in WCCT-1 (CA 0.25% and TSP 0.55% in Wilkins-Chalgren broth), 2.0 × 10⁴/ml in WCCT-2 (CA 0.5% and TSP 0.8% in WC), and 3.3 × 10³/ml in WCCT-3 (CA 1.0% and TSP 2.6% in WC) comparing with 1.3 × 10?/ml in WC after 48 h. Bacteroides fragilis was reduced to 6.0 × 10³/ml in WCCA (CA 0.34% in WC), 2.3 × 10²/ml in WCCT (CA 0.5% and TSP 0.2% in WC), 7.0 × 10³/ml in WCHCl (HCl in WC) after 48 h. Mobiluncus mulieris was reduced to 1.08 × 10⁴/ml in WCCA, 1.03 × 10³/ml in WCCT, and 10 ea/ml in WCHCl after 48 h. Peptostreptococcus asaccharolyticus was completely inhibited in WCCA, WCCT, and WCHCl after 24 h. Probiotics, Steroidobacter denitrificans YH1 (3.4 × 10?/ml) and Lactobacillus crispatus YH2 (2.7 × 10?/ml), grew to 1.25 × 10?/ml and 2.6 × 10?/ml in MRSCA (CA 1.0% in MRS), 1.8 × 10?/ml and 4.6 × 10?/ml in MRSCT (CA 1.5% and TSP 0.58% in MRS), 1.2 × 10?/ml and 2.3 × 10?/ml in MRSHCl after 48 h, respectively. These results mean that the CA-TSP mixture can be used as the useful vaginal pH controller, growth inhibitor on BV causative bacteria, and an efficient means for settlement of probiotics.

The Growth Inhibition Effect on the Causative Bacteria of Bacterial Vaginosis by Bacterial Strains Isolated from the Vagina of a Healthy Woman

Two Gram-positive rod strains isolated from the healthy vagina of a woman were tested for the possibility as probiotics. One strain was identified as Steroidobacter denitrificans (YH1) and the other as Lactobacillus crispatus (YH2) by 16S rRNA partial sequencing. The Casman agar and Man-Rogosa-Sharpe (MRS) agar were mixed in same quantity, supplemented with 5% human rbc lysate (CMS agar). The Wilkins-Chalgren agar and MRS agar were mixed in same quantity (WCM agar). Gardnerella vaginalis was cultured in Casman broth, supplemented with 5% human rbc lysate and 1,000 x-diluted with normal saline. Bacteroides fragilis, Mobiluncus mulieris and Peptostreptococcus asaccharolyticus were cultured in Wilkins-Chalgren anaerobe broth and 2,000x-diluted. S. denitrificans YH1 and L. crispatus YH2 were cultured in MRS broth anaerobically and 100x-diluted. The diluted suspensions of B. fragilis, M mulieris and P. asaccharolyticus were inoculated on WCM agar and G. vaginalis on CMS agar by cotton swabs. Ten ㎕ aliquots of YH1 and YH2 were inoculated on the center of WCM agar and CMS agar. The growth inhibition zone diameters of B. fragilis, G. vaginalis, M. mulieris and P. asaccharolyticus by YH1 were 35 ㎜, 35 ㎜, 25 ㎜ and 60 ㎜. The inhibition diameters by YH2 were 25 ㎜, 30 ㎜, 20 ㎜ and 40 ㎜, respectively. These results implicate that S. denitrificans YH1 can be the stronger probiotics for the treatment of bacterial vaginosis than L. crispatus, compared inhibition zone diameters by YH1 and YH2.

The Growth of <i>Steroidobacter agariperforans</i> sp. nov., a Novel Agar-Degrading Bacterium Isolated from Soil, is Enhanced by the Diffusible Metabolites Produced by Bacteria Belonging to <i>Rhizobiales</i>

An agar-degrading bacterium was isolated from soil collected in a vegetable cropping field. The growth of this isolate was enhanced by supplying culture supernatants of bacteria belonging to the order Rhizobiales. Phylogenetic analysis based on 16S rRNA gene sequences indicated the novel bacterium, strain KA5–BT, belonged to the genus Steroidobacter in Gammaproteobacteria, but differed from its closest relative, Steroidobacter denitrificans FST, at the species level with 96.5% similarity. Strain KA5–BT was strictly aerobic, Gram-negative, non-motile, non-spore forming, and had a straight to slightly curved rod shape. Cytochrome oxidase and catalase activities were positive. The strain grew on media containing culture supernatants in a temperature range of 15–37°C and between pH 4.5 and 9.0, with optimal growth occurring at 30°C and pH 6.0–8.0. No growth occurred at 10 or 42°C or at NaCl concentrations more than 3% (w/v). The main cellular fatty acids were iso–C15:0, C16:1ω7c, and iso–C17:1ω9c. The main quinone was ubiquinone-8 and DNA G+C content was 62.9 mol%. In contrast, strain FST was motile, did not grow on the agar plate, and its dominant cellular fatty acids were C15:0 and C17:1ω8c. Based on its phylogenetic and phenotypic properties, strain KA5–BT (JCM 18477T = KCTC 32107T) represents a novel species in genus Steroidobacter, for which the name Steroidobacter agariperforans sp. nov. is proposed.

Application of a constructed wetland system for polluted stream remediation

Summary In 2010, the multi-function Kaoping River Rail Bridge Constructed Wetland (KRRBW) was constructed to improve the stream water quality and rehabilitate the ecosystem of the surrounding environment of Dashu Region, Kaohsiung, Taiwan. The KRRBW consists of five wetland basins with a total water surface area of 15 ha, a total hydraulic retention time (HRT) of 10.1 days at a averaged flow rate of 14 740 m 3 /day, and an averaged water depth of 1.1 m. The influent of KRRBW coming from the local drainage systems containing untreated domestic, agricultural, and industrial wastewaters. Based on the quarterly investigation results of water samples taken in 2011–2012, the overall removal efficiencies were 91% for biochemical oxygen demand (BOD), 75% for total nitrogen (TN), 96% for total phosphorus (TP), and 99% for total coliforms (TC). The calculated first-order decay rates for BOD, TN, TP, NH 3 –N, and TC ranged from 0.14 (TN) to 0.42 (TC) 1/day. This indicates that the KRRBW was able to remove organics, TC, and nutrients effectively. The high ammonia/nitrate removal efficiency indicates that nitrification and denitrification processes occurred simultaneously in the wetland system, and the detected nitrite concentration confirmed the occurrence of denitrification/nitrification. Results from sediment analyses reveal that the sediment contained high concentrations of organics (sediment oxygen demand = 1.9–5.2 g O 2 /m 2 day), nutrients (up to 15.8 g total nitrogen/kg of sediment and 1.48 g total phosphorus/kg of sediment), and metals (up to 547 mg/kg of Zn and 97 mg/kg of Cu). Appropriate wetland management strategies need to be developed to prevent the release of contaminants into the wetland system. The wetland system caused the variations in the microbial diversities and dominant microbial bacteria. Results show the dominant nitrogen utilization bacteria including Denitratisoma oestradiolicum , Nitrosospira sp., Nitrosovibrio sp., D . oestradiolicum , Alcaligenes sp., Steroidobacter denitrificans , Hydrocarboniphaga effuse were responsible for nitrogen removal, and the dominant carbon degrading bacteria ( Stenotrophomonas maltophilia , H. effuse , Alcaligenes sp., Pseudomonas sp., Fusibacter sp., Chlofoflex i, Guggenheimella bovis , Bacillus pumilus ) were responsible for carbon reduction. The denaturing gradient gel electrophoresis (DGGE) and nucleotide sequence techniques provide a guide for microbial ecology evaluation, which can be used as an indication of contaminants removal. Results from this study show that constructed wetlands have the potential to be developed into an environmentally acceptable river water quality improvement and wastewater polishment alternative for practical application.

Anaerobic and aerobic cleavage of the steroid core ring structure by Steroidobacter denitrificans

The aerobic degradation of steroids by bacteria has been studied in some detail. In contrast, only little is known about the anaerobic steroid catabolism. Steroidobacter denitrificans can utilize testosterone under both oxic and anoxic conditions. By conducting metabolomic investigations, we demonstrated that S. denitrificans adopts the 9,10-seco-pathway to degrade testosterone under oxic conditions. This pathway depends on the use of oxygenases for oxygenolytic ring fission. Conversely, the detected degradation intermediates under anoxic conditions suggest a novel, oxygenase-independent testosterone catabolic pathway, the 2,3-seco-pathway, which differs significantly from the aerobic route. In this anaerobic pathway, testosterone is first transformed to 1-dehydrotestosterone, which is then reduced to produce 1-testosterone followed by water addition to the C-1/C-2 double bond of 1-testosterone. Subsequently, the C-1 hydroxyl group is oxidized to produce 17-hydroxy-androstan-1,3-dione. The A-ring of this compound is cleaved by hydrolysis as evidenced by H2(18)O-incorporation experiments. Regardless of the growth conditions, testosterone is initially transformed to 1-dehydrotestosterone. This intermediate is a divergence point at which the downstream degradation pathway is governed by oxygen availability. Our results shed light into the previously unknown cleavage of the sterane ring structure without oxygen. We show that, under anoxic conditions, the microbial cleavage of steroidal core ring system begins at the A-ring.

Anaerobic testosterone degradation in Steroidobacter denitrificans – Identification of transformation products

The transformation of the androgenic steroid testosterone by gammaproteobacterium Steroidobacter denitrificans was studied under denitrifying conditions. For the first time, growth experiments showed that testosterone was mineralized under consumption of nitrate and concurrent biomass production. Experiments with cell suspensions using [4- 14C]-testosterone revealed the intermediate production of several transformation products (TPs). Characterisation of ten TPs was carried out by means of HPLC coupled to high resolution mass spectrometry with atmospheric pressure chemical ionization as well as 1H and 13C NMR spectroscopy. 3β-hydroxy-5α-androstan-17-one (trans-androsterone) was formed in the highest amount followed by 5 α-androstan-3,17-dione. The data suggests that several dehydrogenation and hydrogenation processes take place concurrently in ring A and D because no consistent time-resolved pattern of TP peaks was observed and assays using 2 TPs as substrates resulted in essentially the same TPs. The further transformation of testosterone in S. denitrificans seems to be very efficient and fast without formation of detectable intermediates.

Initial steps in anoxic testosterone degradation by Steroidobacter denitrificans

Steroid compounds have many important physiological activities in higher organisms. Testosterone and related steroids are important environmental contaminants that disrupt the endocrine systems of animals. The degradation of steroids, especially under anoxic conditions, is challenging because of their complex chemical structure. A denitrifying gamma-proteobacterium, Steroidobacter denitrificans, able to grow anaerobically on a variety of steroids as the sole carbon and energy source was adopted as a model organism to study the anoxic degradation of testosterone. We identified the initial intermediates involved in the anoxic testosterone degradation pathway of S. denitrificans. We demonstrated that under anoxic conditions, S. denitrificans initially oxidizes testosterone to 1-dehydrotestosterone, which is then transformed to androsta-1,4-diene-3,17-dione. In addition, it seems that androst-4-en-3,17-dione can also be directly produced from testosterone by S. denitrificans cells. In general, the initial steps of anoxic testosterone degradation by S. denitrificans are similar to those of the oxic pathway demonstrated in Comamonas testosteroni.

Steroidobacter denitrificans gen. nov., sp. nov., a steroidal hormone-degrading gammaproteobacterium

A denitrifying bacterium, designated strain FS(T), was isolated from anoxic digested sludge on oestradiol [17beta-oestra-1,3,5(10)-triene-3,17-diol] or testosterone (17beta-hydroxyandrost-4-en-3-one) as the sole source of carbon and energy with nitrate as the electron acceptor. Strain FS(T) represents the first known bacterium to grow anaerobically on both oestradiol (C-18) and testosterone (C-19). Steroidal hormones were degraded completely by nitrate reduction to dinitrogen monoxide, which was further reduced to dinitrogen in stationary-phase cultures. Gram-negative cells were slightly curved rods, 0.3-0.5 x 0.6-1.6 microm in size, motile, non-fermentative, non-spore-forming and catalase- and oxidase-positive, showing optimal growth at pH 7.0, 28 degrees C and 0.1% (w/v) NaCl. Beside steroidal hormones, the bacterium utilized only a narrow range of organic substrates with nitrate as the electron acceptor, including several fatty acids and glutamate. No aerobic or anaerobic growth occurred on liquid or solid complex media. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain FS(T) has no known close relatives and represents a distinct lineage within the Gammaproteobacteria. Together with the genera Nevskia, Hydrocarboniphaga, Solimonas and Sinobacter (less than 88% 16S rRNA gene sequence similarity to strain FS(T)), it forms a phylogenetic cluster separated from the families Chromatiaceae, Ectothiorhodospiraceae and Xanthomonadaceae. The quinone system of strain FS(T) consisted exclusively of ubiquinone Q-8. The dominant polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Spermidine in combination with putrescine and traces of sym-homospermidine were the basic polyamines. The major fatty acids detected in testosterone- or heptanoate-grown cells were C(15:0) and C(17:1)omega8c, minor hydroxylated fatty acids were C(11:0) 3-OH and C(12:0) 3-OH. The G+C content of the DNA was 61.9 mol%. Based on the high 16S rRNA gene sequence divergence and different phenotypic properties from previously described gammaproteobacteria in combination with chemotaxonomic data, strain FS(T) is considered to represent a new genus and species, for which the name Steroidobacter denitrificans gen. nov., sp. nov. is proposed. The type strain of Steroidobacter denitrificans is FS(T) (=DSM 18526(T) =JCM 14622(T)).